The Unappreciated Intracellular Lifestyle of Blastomyces Dermatitidis

Published January 14, 2015, doi:10.4049/jimmunol.1303089 The Journal of Immunology

The Unappreciated Intracellular Lifestyle of Blastomyces dermatitidis

Alana K. Sterkel,*,† Robert Mettelman,† Marcel Wuthrich,*€ and Bruce S. Klein*,†,‡

Blastomyces dermatitidis, a dimorphic fungus and the causative agent of blastomycosis, is widely considered an extracellular pathogen, with little evidence for a facultative intracellular lifestyle. We infected mice with spores, that is, the infectious particle, via the pulmonary route and studied intracellular residence, transition to pathogenic yeast, and replication inside lung cells. Nearly 80% of spores were inside cells at 24 h postinfection with 104 spores. Most spores were located inside of alveolar macrophages, with smaller numbers in neutrophils and dendritic cells. Real-time imaging showed rapid uptake of spores into alveolar macrophages, conversion to yeast, and intracellular multiplication during in vitro coculture. The ﬁnding of multiple yeast in a macrophage was chieﬂy due to intracellular replication rather than multiple phagocytic events or fusion of macrophages. Depletion of alveolar macrophages curtailed infection in mice infected with spores and led to a 26-fold reduction in lung CFU by 6 d postinfection versus nondepleted mice. Phase transition of the spores to yeast was delayed in these depleted mice over a time frame that correlated with reduced lung CFU. Spores cultured in vitro converted to yeast faster in the presence of macrophages than in medium alone. Thus, although advanced B. dermatitidis infection may exhibit extracellular residence in tissue, early lung infection with infectious spores reveals its unappreciated facultative intracellular lifestyle. The Journal of Immunology, 2015, 194: 000–000.

lastomyces dermatitidis is the causative agent of blasto- mediated by host immune cells such as neutrophils, macrophages, and mycosis, a potentially deadly fungal infection. The fun- monocytes (11). Moreover, deletion of a global regulator of phase B gus is considered a primary pathogen that can infect transition, dimorphism-regulating kinase 1, locks the fungus in the immune-competent individuals, yet B. dermatitidis can also reactivate mold form and abrogates virulence (12). in previously infected patients that become immunocompromised (1, B. dermatitidis is generally thought of as an “extracellular” 2). B. dermatitidis is one of six dimorphic fungi that are collectively pathogen. Histological sections of infected lung tissue and extra- responsible for most systemic fungal infections in the United States pulmonary sites following fungal dissemination support this premise (3). Infections with the dimorphic fungi represent a growing public by showing that most of the yeast are found in the extracellular health problem, particularly in immunocompromised patients (4), and space. Owing to the long incubation period and the frequent delay in limited measures are available to prevent their acquisition. diagnosing blastomycosis, these data are often collected from human Blastomycosis is commonly reported in endemic regions of patients well after infection is initiated (13–15). This circumstance the United States, Canada, Africa, and the Middle East (5–8). leaves a gap in the knowledge about the early stage of infection. B. dermatitidis, similar to the other dimorphic fungi, grows in the Some evidence points to the intracellular residence of B. der- soil or a similar environmental substrate as a mold, which bears matitidis. Sections of infected tissue have reported yeast inside of spores (conidia). Primary pulmonary infection is initiated when phagocytes (13, 15). In vitro, phagocytes quickly and efﬁciently spores are inhaled into the lungs of a susceptible host (9). There, internalize the small (2–5 mm) spores. The larger yeast (10–30 spores enter alveoli and undergo a morphological transition into bud- mm) are also phagocytosed, but to a lesser extent and at a slower ding yeast. The phase transition to yeast is essential for pathogenesis rate (11). Whereas spores are more vulnerable to killing by phago- of disease (10). Yeast cells are more resistant than spores to killing cytes, yeast can replicate in vitro in their presence, and electron microscopy has revealed multiple yeast inside human monocytes during coculture (16). *Department of Pediatrics, University of Wisconsin School of Medicine and Public Although prior work supports the idea that B. dermatitidis may Health, University of Wisconsin–Madison, Madison, WI 53792; †Department of grow inside of host phagocytes in vitro (16), this and other work Medical Microbiology and Immunology, University of Wisconsin School of Medi- cine and Public Health, University of Wisconsin–Madison, Madison, WI 53792; and did not exclude the possibilities that phagocytes may repeatedly ‡Department of Internal Medicine, University of Wisconsin School of Medicine and internalize yeast from the extracelluar environment, nor that Public Health, University of Wisconsin–Madison, Madison, WI 53792 phagocytes may fuse upon exposure to yeast. Each of these events Received for publication November 14, 2013. Accepted for publication December could also result in the presence of multiple yeast in phagocytes 13, 2014. and give the erroneous conclusion of intracellular replication. This work was supported by National Institutes of Health Grant AI-035681 and by an American Fellowship from the American Association of University Women. Additionally, the in vitro studies above were done with yeast and not spores, the infectious particles that initiate infection. If spores Address correspondence and reprint requests to Dr. Bruce S. Klein, University of Wisconsin, Microbial Sciences Building, 1550 Linden Drive, Madison, WI 53706. are indeed rapidly internalized and highly sensitive to killing by E-mail address: [email protected] leukocytes, this raises the question of where and how inhaled The online version of this article contains supplemental material. spores convert into yeast and replicate during early infection to Abbreviations used in this article: BAD-1, blastomyces adhesin 1; BMM, bone establish disease. To our knowledge, studies of pulmonary blas- marrow macrophage; DC, dendritic cell; DTR, diphtheria toxin receptor; DTx, diph- tomycosis have not been conducted with spores to initiate lung theria toxin; WT, wild-type. infection and interrogate their intracellular residence, transition, Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 and replication during the early pathogenesis of disease.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303089 2 INTRACELLULAR LIFESTYLE OF B. DERMATITIDIS

In this study, we investigated the early pathogenesis of pul- system. Spores were delivered intratracheally through a cannula in a 20 ml monary blastomycosis in a model involving infection with spores. suspension in PBS. We investigated the host–pathogen interaction with emphasis on Flow cytometry elucidating intracellular residence and replication of the fungus. m We tackled several questions. First, are spores taken into phago- Lungs were diced by pressing them through a 40- m filter with the plunger of a 5-ml syringe. Homogenates were digested using 1 mg/ml collagenase cytes in the lung, and, if so, what are the cells and time course? D (Roche) and 10 ng/ml DNAse I (Sigma-Aldrich) for 20 min at 37˚C. Second, do spores convert to yeast inside lung phagocytes? Third, RBCs were lysed with ammonium chloride/potassium bicarbonate buffer do yeast replicate inside these cells, and to what extent are mul- and the remaining cells were washed with 2 mM EDTA and 0.5% BSA in m tiple intracellular yeast due to replication, multiple phagocytic PBS (FACS buffer). Staining was done in 100 l FACS buffer for 20 min at 4˚C in the dark. Stained cells were washed with FACS buffer, fixed with events, or cell fusion? Finally, if B. dermatitidis replicates inside 2% paraformaldehyde solution for 20 min, washed again, and suspended in host lung phagocytes, do the phagocytes constrain or permit FACS buffer for analysis. All centrifugation was done at 1500 rpm for progression of early infection? We report that spores are rapidly 5 min and 4˚C in an Eppendorf 5825 centrifuge. Events were gated on taken up into alveolar macrophages where they convert to yeast forward light scatter and side light scatter to exclude debris and Live/Dead fixable yellow stain (Molecular Probes) was used to exclude dead cells. and replicate intracellularly. Moreover, intracellular residence and + + Alveolar macrophages were defined phenotypically as CD11c , Mac3 , replication in macrophages is required for initiation of disease. CD11b2, CD1032, Ly6g2, and autofluorescent. Neutrophils were defined as CD11b+, Ly6g+ (clone 1A8), and CD11c2. Dendritic cells (DCs) were Materials and Methods defined as CD11c+, MHC class II+ with the following subsets: neutrophil- derived DCs were CD11c+, CD11b+, and Ly6g+; inflammatory DCs were Mice 2 CD11c+,CD11b+,andLy6g ; and resident DCs were CD103+ and + C57BL/6 wild-type (WT) mice were obtained from the National Cancer CD11c (19–21). Abs were conjugated to the fluorophores FITC, PE, Institute. CD45.1 C57BL/6 mice were obtained from Taconic. CD11c– PerCP, PE-Cy7, allophycocyanin, Alexa Fluor 700, or allophycocyanin- diphtheria toxin (DTx) receptor (DTR) mice were obtained from The Cy7 and were from BD Biosciences, eBioscience, and BioLegend. Jackson Laboratory and bred in-house. Mice were housed and cared for Spores and yeast were identified in FACS by low and high expression, according to guidelines from the University of Wisconsin Animal Care and respectively, of mCherry fluorescence as noted above and illustrated in Use Committee, who approved this work. Their guidelines are in com- Results. Extracellular fungi were stained with 10 mg/ml Uvitex 2B (Poly- pliance with Health and Human Services Guide for the Care and Use of Sciences). The percentage of fungi that were intracellular was defined as + 2 Laboratory Animals. the number of mCherry /Uvitex 2B events divided by the total number of fungi that stained with either dye. Data were collected on an LSR II Reagents and cell culture cytometer (BD Biosciences) and analyzed by FlowJo software (Tree Star). Bone marrow was collected to generate bone marrow–derived macrophages Microscopy (BMMs) and chimeric mice. Marrow was collected from femurs and tibias by rinsing and disruption through a 26-gauge needle and filtering via To characterize yeast inside of AMJ2-C11 macrophages, cells were cultured a 40-mm filter. BMMs were differentiated in culture at 37˚C using a 1:10 for 24 h with 26199 yeast in a 24-well plate with cRPMI. The medium was dilution of L929 supernatant, and adherent cells were collected after 1 wk. removed and cells were stained with 10 mg/ml Uvitex 2B to identify ex- Alveolar macrophages were collected from exsanguinated mice by re- tracellular yeast and 12 mM ethidium bromide to identify dead cells in peated lavage of the lung alveoli through a cannula with 0.6 mM EDTA in PBS. Washed cells were fixed with 2% paraformaldehyde and per- PBS at 37˚C. The lavage fluid was then placed on ice. RBCs were lysed meabilized with 0.05% saponin (Sigma-Aldrich) for 20 min. Cells were using ammonium chloride/potassium bicarbonate buffer. The murine stained with anti–BAD-1-FITC to identify yeast. Anti–BAD-1 mAb (DD5- alveolar-like macrophage cell line AMJ2-C11 was obtained from the CB4) (22) was made from ascites, ammonium sulfate precipitated, purified American Type Culture Collection. Cells were counted on a hemocytom- on an A/G agarose column (Pierce Chemical), and then labeled with FITC eter using trypan blue (Sigma-Aldrich) to assay viability. Cells were (Molecular Probes) and purified by dialysis. Differential fluorescence cultured at 37˚C and 5% CO2 and maintained in RPMI 1640 medium microscopy was performed with an Olympus BX60 microscope. Images (HyClone) with 10% heat-inactivated (56˚C for 30 min) FBS (Atlanta were captured with an EXi Aqua Camera (QImaging) and QCapture Pro Biologicals), 100 U penicillin, and 100 mg streptomycin (HyClone). 6.0 image software. Images were processed using Adobe Photoshop. Live imaging was done on an Observer Z1 microscope with a humidified in- Fungi cubation chamber kept at 37˚C and 5% CO2, and data were collected using AxioVision software (Zeiss). Videos were processed using iMovie. B. dermatitidis yeast from strains 26199 and 14081 (American Type Culture Collection) were taken in log-phase growth and suspended in PBS. Intracellular replication Yeast were aspirated through a 26-gauge needle and passed through a 5 40-mm filter to reduce aggregated yeast. In some experiments, yeast were In vitro intracellular replication was quantified by culturing 7 3 10 BMMs 4 heat-killed at 65˚C for 1 h in PBS. Yeast CFU were counted from brain or alveolar macrophages with 7 3 10 yeast at a multiplicity of infection of heart infusion plates after 5–7 d of growth at 37˚C. Yeast from strain 14081 0.1 on glass coverslips in a 24-well plate. We used yeast that express either were plated onto potato dextrose agar and incubated for two wk at 22˚C. red (mCherry) or green (GFP) fluorescence for these assays. We added the The resulting hyphal mat was lightly rubbed with a spreader and 5 ml PBS yeast at a 1:20 ratio of red to green yeast to reduce the likelihood of to collect spores. The resulting suspension was filtered through multiple multiple yeast within a single macrophage arising from multiple phago- layers of sterile Miracloth (Millipore) and a 40-mm filter to reduce hyphal cytic events. After allowing the yeast to be phagocytosed for 4 h, free yeast contamination (,2% in all experiments). Spore CFU were counted from were washed away with PBS. At 4, 24, 48, and 72 h time points, samples Kelly’s agar plates incubated at 30˚C for 8–10 d. All work with spores was were collected by washing the coverslips with PBS and staining with either performed under biosafety level 3 conditions. Live/Dead fixable violet stain (Molecular Probes) as per the manu- Fluorescent yeast were used in some experiments. A yeast strain facturer’s instructions, or with 10 mg/ml Uvitex 2B for 20 min to exclude expressing GFP under the control of a constitutive, histone H2B promoter extracellular yeast from analysis. Cells were then fixed using a 2% para- has been described (17). We used mCherry fluorescent protein under the formaldehyde solution for 20 min, washed with PBS, and stationed onto control a yeast phase-specific blastomyces adhesin 1 (BAD-1) promoter a glass slide cell face down in Mowiol 4-88 anti-fade medium (Calbio- (18) to create a “reporter” strain of 14081 in which spores fluoresced dimly chem). Wells that were not collected at a time point were washed to and phase transition led to intense expression and bright red yeast via remove extracellular yeast and replenished with fresh media. Fixed slides fluorescence microscopy or FACS. Thus, a dim mCherry signal was suf- were maintained in the dark at 4˚C until imaging. ficient to allow tracking of spores in vivo by FACS, and the increased signal of .10-fold was used to confirm phase transition to yeast (see Macrophage fusion Results). BMMs were stained with 15 mM 5-chloromethylfluorescein diacetate Infection (Invitrogen) or PKH26 (Sigma-Aldrich), as per the manufacturers’ instructions, and equal parts of the stained cells were aliquoted into a 24- Mice were anesthetized with isoflurane and suspended by their front incisors well plate containing glass coverslips for a total of 7 3 105 cells per well, from a wire on a 45˚ plane. Mice were intubated with a BioLite intubation as described above for quantification of intracellular replication. Fusion The Journal of Immunology 3 was stimulated with 7 3 104 yeast or, as a positive control, with the ad- were treated with DTx and anti-Ly6g 1 d before infection and lungs were dition of 10 ng/ml IL-4 (PeproTech) and 1 mg/ml GM-CSF (R&D Sys- collected 2 d postinfection. tems) (23). Statistical analysis Bone marrow chimeras Experimental conditions were compared with controls using an unpaired Administration of DTx (Sigma-Aldrich) to CD11c-DTR mice is lethal after Student t test, a Mann–Whitney U test, or ANOVAwith a Tukey’s multiple more than two treatments of 100 ng (see Fig. 4). Therefore, we generated comparison test where appropriate. A p value ,0.05 was considered bone marrow chimeric mice as previously described (24). Briefly, chimeric statistically significant. Analysis was performed with Prism software mice were generated by lethally irradiating CD45.1 mice with two doses of (GraphPad Software). Data are presented as means, and error bars 550 rad 4 h apart on an X-RAD 320 (Precision X-ray). Mice were injected represent SEM. with 1 3 107 bone marrow cells i.v. from either CD45.2 WT or CD11c- DTR donor mice. Chimeric mice were maintained on 0.5 mg/ml Baytril 100 (Bayer) and 2 mg/ml neomycin sulfate (Sigma-Aldrich) in their Results drinking water for 2 wk. Alveolar macrophages were allowed to recon- Interaction between spores and leukocytes early during stitute for 12 wk. Mice were injected with 100 ng DTx i.p. every other day infection starting 2 d before infection with 3.2 3 104 spores. Lungs were collected every 2 d for 12 d and analyzed by FACS and for CFU. Pulmonary infection with B. dermatitidis begins with inhalation of infectious particles. Thus, we introduced spores into the lungs of Neutrophil depletion mice to mimic the natural infection. Alveolar macrophages high 2 + Neutrophils were depleted by i.v. injection every other day with 250 mg (CD11c , CD11b ,Mac3) are the predominant leukocyte anti-Ly6g (clone 1A8; Bio X Cell). Rat IgG was used as a control. Mice present in the lungs of naive mice, with 5 3 105 cells, and they

FIGURE 1. Most spores reside in alveolar macrophages early during infection. Mice were infected with mCherry 14081 spores by intubation. (A)At24h postinfection, lung homogenates were analyzed for the total number and lineage of leukocytes. The gating markers and strategy are described in Materials and Methods and illustrated in Supplemental Fig. 1. AM, alveolar macrophages; Neu, neutrophils. DC subsets (inlay) are resident (Res), neutrophil-derived (Neu), and inflammatory (Inflam). (B) The proportion of total spores (mCherry+ events) in the lungs that are intracellular (Uvitex 2B2) at 1 and 24 h postinfection was measured at a range of inocula. Results are representative of three experiments, each with five mice per group. (C) Intracellular residence of spores within alveolar macrophages from lung homogenates of mice infected with mCherry spores for 12 h. Alveolar macrophages were identified with anti–CD11c-FITC. Uvitex 2B stain of chitin was used to determine whether the spores were extracellular (only extracellular spores bind the dye). Scale bar, 25 mm. (D) Distribution of intracellular spores (mCherry+, Uvitex 2B2) 24 h after mice were infected with 1 3 105 spores. A flow plot of a representative mouse illustrating intracellular spores (red dots) overlaid on total leukocytes (gray) with representative gates for alveolar macrophages (top left), DCs (top right), and neutrophils (bottom right) is shown. Distribution of spores within leukocytes (left panel) is based on the gating strategy shown in Supplemental Fig. 1; group mean 6 SEM was analyzed with FACS (right panel). (E) The total number of lung leukocytes that contain spores 24 h postinfection. Results shown in (D) and (E) are representative of three experiments with five mice per group. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. 4 INTRACELLULAR LIFESTYLE OF B. DERMATITIDIS also accounted for most of the leukocytes in the lungs early during analyzed whole-lung homogenates by flow cytometry (Fig. 1D). infection (Fig. 1A, Supplemental Fig. 1). Infection with spores in- Independent of the inoculum, most (.70%) intracellular spores duced inflammation and the influx of other leukocytes (Fig. 1A) were detected in alveolar macrophages after 24 h of infection, compared with naive mice, which have 1 3 104 neutrophils with only minor proportions in DCs and neutrophils (Fig. 1D). (CD11b+, CD11c2, Ly6g+)and53 104 DCs (CD11c+, MHC class The selective association of spores with alveolar macrophages II+) in their lungs (data not shown). Higher inocula induced a more remained strong even at a higher inoculum of 105 spores, which prominent influx of these cells. Infection with spores also led to the leads to greater numbers of neutrophils in the lung. At 105 spores, influx of a rarely described DC subset, neutrophil-derived DCs there were ∼4700 alveolar macrophages with intracellular spores (CD11c+, CD11b+, Ly6g+) (Fig. 1A, inset, Supplemental Fig. 1), versus only 700 neutrophils and 250 DCs (Fig. 1E). Thus, most which are barely detected in naive mice (25). B. dermatitidis spores enter leukocytes early during infection and We used B. dermatitidis that expresses mCherry protein fluo- these spores reside predominantly within alveolar macrophages, rescence to let us track (dimly fluorescent) spores in the lungs of not neutrophils or DCs. infected mice. We analyzed the intracellular residence of spores by identifying extracellular spores with the membrane-excludable, Fate of intracellular spores and yeast chitin-specific stain Uvitex 2B. We found a dose-dependent effect The high percentage of spores found inside alveolar macrophages on intracellular residence, with lower spore inocula showing more suggests a likely role for macrophages early during infection, either intracellular infection than did higher inocula (Fig. 1B). The lower in restricting growth of the fungus or providing a locale for in- limit of reliable detection by FACS was with an inoculum of 104 tracellular replication and establishment of infection. We therefore spores. At this inoculum, 70–80% of spores were located inside investigated the ability of the fungus to survive, convert from spores leukocytes by 24 h postinfection. Although spores could not be to yeast, and replicate in alveolar macrophages. To monitor the reliably tracked at a lower inoculum, intracellular residence in- phase transition of spores to yeast, we exploited the B. dermatitidis creased as the inoculum was reduced and thus could be higher at reporter strain. In this strain, the yeast phase–specific gene pro- inocula of ,104 spores. moter (BAD-1) upregulates mCherry fluorescence during the We sought to identify the cells in which spores reside early phase transition from mold or spore to yeast (18). Hyphae have no during infection. Spores were readily identified inside alveolar fluorescence, spores show dim expression, and yeast highly ex- macrophages upon microscopic analysis of homogenized lung press mCherry (Fig. 2A). Spores of this strain were rapidly in- tissue (Fig. 1C). To quantify the distribution of spores, we further ternalized by alveolar macrophages during in vitro coculture, with

FIGURE 2. B. dermatitidis spores survive and replicate as yeast in alveolar macrophages. (A) Bright- field and fluorescence microscopy of spores and yeast of reporter strain 14081 that upregulates mCherry under the control of a yeast phase–specific BAD-1 promoter. mCherry fluorescence is quantified by FACS in Fig. 6A. (B) Uptake of mCherry spores by macrophages in vitro. Spores were cultured with primary alveolar macrophages at a multiplicity of infection (MOI) of 0.2. Uptake was quantified during 4 h and analyzed by FACS. Extracellular spores stained Uvitex 2B+. Results are the means of triplicate wells and are representative of three experiments. (C) Live imaging of mCherry 14081 spores cultured with primary alveolar macrophages at an MOI of 0.2. Images shown are every 5 h. The full video is available online. (D) Mice were infected with mCherry 14081 spores and 3 d later bronchoalveolar lavage fluid was collected and stained with anti–CD11c-FITC to identify alveolar macrophages and Uvitex 2B to distinguish intracellular and extracellular yeast. Inlay in the left panel shows a budding yeast cell overlying another yeast. (E) Al- veolar macrophage cell line AMJ2-C11 was cultured for 48 h with 14081 yeast on coverslips. Cultures were treated with Uvitex 2B to stain the extracellular yeast, and with ethidium bromide to ascertain macrophage membrane integrity and cell viability. Cultures were fixed and then permeabilized to identify yeast with anti–BAD-1-FITC. Scale bars, 25 mm. The Journal of Immunology 5

95% of spores inside macrophages by 2 h (Fig. 2B). During live red yeast, we greatly increased the probability that multiple, cell imaging of these spores cultured with primary alveolar intracellular red yeast arose from replication in macrophages macrophages, we observed intracellular transition of the spores to and not from multiple phagocytic events or macrophage fusion yeast and ensuing replication (Fig. 2C, Supplemental Video 1). (Fig. 3A). For example, the probability that four red yeast in We also detected budding yeast within individual alveolar mac- a macrophage arose from multiple phagocytic events is low, that rophages found in lavage fluid from spore-infected mice (Fig. 2D), is, ∼1/1.6 3 105. Briefly, we cultured BMMs with yeast in vitro consistent with intracellular replication in vivo. Moreover, when for 4 h to enable phagocytosis and then removed the nonadherent yeast were cultured with AMJ2-C11 macrophages in vitro, some yeast. We incubated cultures for an additional 24 h to allow yeast macrophages contained 30 or more yeast after 2 d of culture (Fig. to replicate. Then, we counted the number of red yeast inside 2E). Thus, spores can survive, germinate, and even replicate as macrophages. We excluded extracellular yeast that stained posi- yeast inside of alveolar macrophages shortly after infection. tive for Uvitex 2B and macrophages that contained green yeast. Macrophages cultured with live yeast had more red yeast per Intracellular replication of yeast inside macrophages macrophage than did macrophages cultured with heat-killed yeast Because B. dermatitidis is not considered a facultative intracel- (Fig. 3B). Because only cocultures with live yeast resulted in three lular pathogen that replicates inside macrophages, we further in- or more red yeast per macrophage (cultures with heat-killed yeast vestigated the extent to which replication occurs intracellularly. had only one to two red yeast per macrophage), we concluded that The finding of multiple yeast in a single macrophage could be the three or more yeast per macrophage was likely due to intracellular result of multiple phagocytic events, fusion of macrophages, or replication, not multiple phagocytic events. Some macrophages replication of yeast inside of a macrophage. To maximize the had seven or more intracellular red yeast. The percentage of likelihood of quantifying only intracellular replication events, we BMMs that contained three or more red yeast grew steadily over cocultured macrophages in vitro with an inoculum containing red time during 72 h of incubation (Fig. 3C). Similar trends were (red fluorescent protein) and green (GFP) yeast in a ratio of 1:20. observed in cocultures of live yeast with primary alveolar mac- By interrogating macrophages that harbor only the less common rophages (data not shown). The increasing proportion of macro-

FIGURE 3. Intracellular replication by B. dermatitidis yeast. (A) Probability of only red yeast being phagocytosed when they comprise 5% of the yeast inoculum. (B) BMMs were cultured for 24 h with two yeast strains, one expressing red fluorescent protein and the other, GFP (ratio of 1:20, respectively); both strains in an assay were either live or heat-killed. The number of yeast per macrophage was enumerated for the BMMs that contained only red yeast. Five hundred yeast-containing macrophages were enumerated per condition; results are the means 6 SEM of five experiments. (C) BMMs containing three or more red yeast were enumerated. Five hundred yeast-containing macrophages were counted per time point; results are the means 6 SEM of three experiments. (D) The fusion of a red macrophage with a green one results in a yellow, multinucleated giant cell. (E) Fusion between PKH26 (red)- stained and 5-chloromethylfluorescein diacetate (green)–stained BMMs (1:1) was enumerated for cells cultured 24 h with or without yeast (strain 26199) or with IL-4 and GM-CSF (control). Results are the means 6 SEM of four experiments in which .400 macrophages were counted per condition. (F) A fused, yellow, multinucleated giant cell from a culture with yeast that does not contain intracellular yeast. (G) Red and green BMMs were cultured with yeast over time. The proportion of fused BMMs with three or more intracellular yeast is depicted. Results are the means 6 SEM of three experiments in which .400 macrophages were counted per condition. *p , 0.05, ***p , 0.001. 6 INTRACELLULAR LIFESTYLE OF B. DERMATITIDIS phages with three or more yeast over time suggests that yeast DTx to survive multiple doses (Fig. 4A). Thus, we created bone frequently replicate inside macrophages. marrow chimeric mice in which WT CD45.1 mice were lethally The ratiometric analysis above reduced the chance that repeated irradiated and reconstituted with bone marrow from CD45.2 WT or phagocytic events could account for the finding of multiple in- CD45.2 CD11c-DTR mice (Fig. 4B) (24). Chimeric CD11c-DTR tracellular red yeast, but fusion of macrophages could not be en- mice tolerated multiple doses of DTx, similar to WT mice tirely excluded. We therefore assayed whether yeast induce fusion (Fig. 4A). Although CD11c is expressed on alveolar macrophages of macrophages and the extent to which this event explained our as well as DCs, toxin treatment reduced the macrophages by 21-fold finding of multiple yeast inside a macrophage. In this assay, BMMs and the DCs by only 2.6-fold (Fig. 4C). Depletion of CD11c+ cells were stained with red or green fluorescent dye and mixed together in chimeric mice that were challenged with B. dermatitidis spores in equal proportions; fusion of the two yielded a yellow, multi- resulted in a surprising 26-fold reduction in CFU by day 6 postin- nucleated giant cell (Fig. 3D). Yeast did induce macrophage fu- fection, compared with WT chimeric mice treated with DTx. This sion, but it occurred in only 2% of macrophages (Fig. 3E). This trend was seen across multiple time points (Fig. 4D). Toxin treat- amount of fusion was similar to that found for BMMs exposed to ment of WT mice and DTR expression in the absence of toxin IL-4 and GM-CSF, which are known to induce fusion (26). treatment did not independently reduce lung CFU (Fig. 4D). Thus, Nevertheless, not all fused macrophages harbored intracellular CD11c+ cells in the lung were essential for propagation and path- yeast (Fig. 3F). In fact, fusion accounted for only a minority ogenesis of infection, and their elimination unexpectedly stemmed (#20%) of BMMs with three or more yeast (Fig. 3G). Thus, al- the extent of pulmonary disease after infection with spores. though coculture with B. dermatitidis induces macrophage fusion, Role of neutrophils during spore infection the phenomenon does not contribute substantially to the finding of multiple yeast inside macrophages. Although we show above that yeast can replicate inside alveolar macrophages, it is also possible that residence inside these Role of macrophages during progressive pulmonary infection phagocytes protects the fungus from other more potent leukocyte Because we observed that most of the spore inoculum entered effectors. Neutrophils are more effective than macrophages at alveolar macrophages early during infection, and that these par- killing B. dermatitidis spores and yeast (11, 27). Neutrophils also ticles convert to yeast that replicate intracellularly, we sought to comprise the next largest population of leukocytes (after alveolar distinguish whether alveolar macrophages are required to constrain macrophages) in the lungs of spore-infected mice (Fig. 1A). We the early infection or, alternatively, enable replication and pro- also observed that the proportion and number of lung neutrophils gression of infection. We sought to use CD11c-DTR mice to ad- increased significantly after infection in toxin-treated versus un- dress the role of macrophages in the pathogenesis of early infection. treated control mice (Fig. 5A, 5B). This neutrophilia upon toxin We found that uninfected CD11c-DTR mice are too sensitive to treatment is consistent with previous work with these mice (28).

FIGURE 4. Lung CD11chigh cells permit progressive pulmonary infection. (A) Uninfected mice were injected with 100 ng DTx i.p. every other day for up to 12 d. Results are representative of five independent experiments. (B) Lung homogenates of bone marrow chimeric recipients (CD45.1) that received congenic (CD45.2) WT control or CD11c- DTR bone marrow were assayed by flow cytometry for reconstitution of hematopoietic cells. Results are the means 6 SEM of two experiments with 5–18 mice per group. (C) The efficiency of CD11c+ cell depletion in DTx-treated mice after reconstitution with WT or CD11c-DTR bone marrow. The percentage of alveolar macrophages and DCs and the absolute cell numbers following depletion are shown in flow plots (left) and a bar graph (right). Results are representative of three experiments with five mice per group. (D) Lung CFU of DTx- or PBS control–treated chimeric WT and CD11c-DTR mice 6 and 10 d postinfection with mCherry 14081 spores. Results are representative of two experiments with three chimeric mice per group. *p , 0.05, **p , 0.01, ****p , 0.0001. The Journal of Immunology 7

Toxin-treated CD11c-DTR mice showed a .6-fold increase in the Effect of macrophages on phase transition of spores to yeast percentage of leukocyte-associated spores that were associated Because spores are more vulnerable than yeast to killing by leu- with neutrophils (9 versus 56%) (Fig. 5D, left panel); numbers kocytes (11), the rapidity of phase transition would likely offer showed corresponding trends (Fig. 5D, right panel). a selective advantage to the fungus in survival early during in- We hypothesized that if macrophages offer a relatively “pro- fection. By flow cytometric analysis, transition reporter yeast tected” locale for spores against neutrophils, then an increased display a 10-fold higher mCherry fluorescence than do spores exposure to neutrophils in toxin-treated mice might explain the (Fig. 6A). Using this reporter strain during infection, we found reduced lung CFU after depletion of CD11c+ cells. To test this that the phase transition of spores to yeast is significantly delayed hypothesis, we depleted neutrophils with an Ly6g-specific Ab in in the first week of infection in chimeric mice that are depleted of toxin-treated CD11c-DTR mice before infection with spores CD11c+ cells (Fig. 6B); this delay in transition correlated with the (Fig. 5A, 5B). Neutrophil depletion did not affect the number of time frame in which lung CFU are sharply curtailed in these mice. alveolar macrophages (compared with rat IgG treated controls) We also used the reporter strain for in vitro studies with mac- (Fig. 5C). Whereas the lung CFU postinfection rose slightly in rophages. The percentage of spores that demonstrated phase neutrophil-depleted WT mice versus untreated mice, lung CFU transition to yeast in vitro during 4 d nearly tripled during coculture did not increase significantly after neutrophils were depleted from with BMMs (Fig. 6C) and alveolar macrophages (data not shown) toxin-treated CD11c-DTR mice (Fig. 5E). Spores did show compared with culture in medium alone. During this time frame, a small increase in association with DCs in toxin-treated CD11c- little to no replication of the fungus occurred in vitro as deter- DTR mice that were depleted of neutrophils (Fig. 5F). Thus, ex- mined by CFU analysis (data not shown). These data indicate that posure of B. dermatitidis to neutrophils did not appear to explain infectious spores undergo significantly faster transition to patho- + the sharply reduced lung CFU in mice depleted of CD11c cells, genic yeast in vivo in the lungs of mice in which CD11c+ cells, that is, chiefly alveolar macrophages. This implies that B. der- that is, mainly alveolar macrophages, are present. Spores also matitidis spores may benefit directly from the growth environment evince more rapid transition to yeast when they are cocultured inside macrophages. in vitro with macrophages.

FIGURE 5. Neutrophils do not account for reduced lung CFU in CD11c-depleted mice. CD11c-DTR mice were treated with rat IgG, 100 ng DTx i.p., anti-Ly6g Ab i.v., or both DTx and Ab. Mice were infected with mCherry 14081 spores and lung homogenates were analyzed 2 d later. (A) Flow plots showing the proportion of lung neutrophils in representative mice. (B) Neutrophil numbers were quantiﬁed by FACS and hemocytometer count. (C) The number of alveolar macrophages (AM) in mice given DTx alone or together with neutrophil-depleting Ab. (D) Flow plot of the distribution of intracellular spores (mCherry+, Uvitex 2B2) denoted as black dots with respect to leukocytes (gray) and alveolar macrophages (AM; top left gate), DCs (top right), and neutrophils (Neu; bottom right) in representative PBS- versus toxin-treated CD11c-DTR mice (left panel). The right panel shows the number of intracellular spores associated with each cell type. (E) Lung CFU in mice corresponding to (A)– (C). (F) The distribution of spores among leukocytes in depleted or control mice evaluated by FACS. All results are representative of three independent experiments with ﬁve mice per group. *p , 0.05, **p , 0.01, ****p , 0.0001. 8 INTRACELLULAR LIFESTYLE OF B. DERMATITIDIS

FIGURE 6. Macrophages accelerate phase transition of spores to yeast. (A) Flow cytometric analysis of mCherry expression on spores and yeast. (B) Chimeric WT and CD11c-DTR mice treated with DTx in Fig. 4 were infected with mCherry 14081 spores. The percentage of B. dermatitidis that underwent transition from spore to yeast in lung homogenates was defined as the proportion of mCherry events that were brighter than the threshold defined by FACS in (A). The inset shows the CFU in these mice on the same time scale. Results are the means 6 SEM of three mice per group and are representative of two experiments. (C) mCherry 14081 spores were cultured in vitro in medium alone or with BMMs at a multiplicity of infection of 0.1 for 4 d and analyzed by flow cytometry for intensity of mCherry expression. Expression of mCherry beyond the dotted line was defined as transition to the yeast phase defined by FACS in (A). Results in the left panel are those of representative wells. The right panel shows the percentage of events that were mCherry high (yeast) quantified and averaged among triplicate wells. Results are the means 6 SEM and are representative of three experiments. ***p , 0.001, ****p , 0.0001.

Discussion lication inside primary alveolar macrophages. Based on our find- B. dermatitidis is generally considered an extracellular pathogen. ings that three or more yeast within a single macrophage is most Histological sections of infected patient tissues typically show likely due to replication, and not multiple phagocytic events or a high proportion of the yeast in the extracellular space. However, macrophage fusion, our finding of three or more yeast and budding most data from patients represent the late stage of infection once within single alveolar macrophages from the bronchoalveolar la- large numbers of yeast occupy tissue and neutrophils dominate. vage fluid of infected mice supports the premise that intracellular The large size of budding yeast can be hard for neutrophils to replication occurs in vivo. ingest, perpetuating the notion of extracellular residence. The role Previous investigators have sought to evaluate intracellular of intracellular residence in early pathogenesis and initiation of replication of B. dermatitidis yeast within macrophages in vitro infection with spores has not been investigated. (16, 33). Their findings of an increase in CFU may have been In this study, using a murine model of infection with spores, we confounded by the potential growth of extracellular or partially observed that a high proportion of the spores that reach the lungs internalized yeast. Our live imaging data demonstrate unequivocal are located inside of alveolar macrophages within 24 h of infection. intracellular replication by yeast, and they are further supported by Alveolar macrophages contained most of the spores after pulmo- our ratiometric studies. Using quantitative analysis involving red nary infection despite a rapid influx of neutrophils, inflammatory and green yeast at a defined ratio of 1:20, we established that the DCs, and even neutrophil-derived DCs. This finding contrasts with increasing number of live yeast per macrophage was due to in- reports of late-stage infection dominated by yeast and pyogranu- tracellular replication over time. We chose the ratiometric method lomas (13). We observed that the lowest inoculum yielded the of investigation over an alternate method of measuring the bud- greatest association of spores with alveolar macrophages, ding index, where the proportion of budded yeast is quantified to approaching 80%. Although our flow cytometric analysis was identify growth under different conditions (34–36). We found that not sensitive enough to reliably detect leukocyte interactions regardless of the intracellular or extracellular location of yeast, with ,104 spores, others have reported that as few as 70 spores a high proportion of them (.70%) are budding (data not shown). are sufficient to cause disease and death in mice (29). Although This prevented us from observing a change in the proportion of the inoculum of spores that causes disease in humans is unknown, yeast that bud under different conditions and using the budding our findings demonstrate that at low inocula most of the infectious index to evaluate intracellular replication. spores enter alveolar macrophages, a niche in which they undergo We considered the possibility that our quantitation of intracel- morphogenesis to initiate infection and suppress innate host de- lular replication could be confounded by cell fusion and formation fense such as NO and TNF-a production (30, 31). of giant cells. Multinucleated giant cells are observed during We used live cell imaging to investigate the uptake of spores by histological analysis of B. dermatitidis–infected human lungs. naive alveolar macrophages, phase transition to yeast and intra- These cells are commonly seen in proximity to yeast and occa- cellular replication in real time. Although spores are vulnerable to sionally with intracellular yeast (14). We did find evidence that killing by macrophages, and infection has been thought to arise yeast induce macrophage fusion and multinucleated giant cells. from extracellular spores (32), we found that .95% of spores were However, this was a rare occurrence and did not alter our con- taken up by 2 h of incubation, and that spores converted to yeast, clusions because the vast majority of macrophages with three or which replicated inside alveolar macrophages. Although we are more yeast were not fused. Furthermore, ratiometric analysis re- currently unable to monitor spore transition in vivo in real time, duced the likelihood that fusion confounded our analysis: a mac- our imaging in vitro established intracellular transition and rep- rophage containing a red yeast was 20-fold more likely to fuse The Journal of Immunology 9 with a macrophage containing a green yeast than one with another In summary, we provide new insight about the early stages of red yeast, and macrophages that contained the more prevalent B. dermatitidis infection after inhalation of spores. Although in- green yeast were excluded from analysis. tracellular residence and replication appear to be integral parts of In view of finding most spores within alveolar macrophages early infection, the extent to which such events play a role in late in vivo, and strong in vitro evidence of intracellular phase transition infection remain unknown. Intracellular residence could enable and replication, we tested whether macrophages constrain or fa- B. dermatitidis to spread from the lungs to extrapulmonary tissue. cilitate early pathogenesis of infection. CD11c-DTR mice were This “Trojan Horse” method of dissemination has been proposed useful to address this question, although the intolerance of these for other intracellular fungi such as C. neoformans (41–45), where mice to multiple toxin treatments required the generation of chi- in a murine model of cerebral infection yeast have been detected meric mice. Depletion of CD11chigh cells sharply curtailed lung in monocytes circulating in blood and in leptomeningeal capil- CFU in the toxin-treated chimeric mice. We interpret this finding laries of brain sections. New insight into how B. dermatitidis as evidence that the entry of spores into alveolar macrophages grows in host cells may likewise improve our understanding of permits initiation of infection. We sought to restore lung macro- how the fungus establishes itself in the lung and disseminates to phages by adoptive transfer back into depleted mice, but we were visceral sites. unsuccessful in reconstituting the numbers; thus, we lack formal proof for the role of alveolar macrophages per se. Cells other than Acknowledgments alveolar macrophages express CD11c, for example DCs, and they We thank Lori Neal for advice, Robert Gordon for assistance with graphic are also depleted in CD11c-DTR mice. However, DCs are unlikely illustrations, and Tom Sullivan and Hugo Paes for engineering the mCherry to account for the reduction in CFU we observed. There are many reporter strain of B. dermatitidis. more alveolar macrophages in the lungs and spores chiefly associated with them and not DCs. Moreover, DTx was 10-fold more Disclosures efficient in depleting alveolar macrophages than DCs. 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